Construction, programming and testing of measurement ...1078863/FULLTEXT01.pdf · bacteria growth,...

UPTEC F 16062

Examensarbete 30 hpFebruari 2017

Construction, programming and testing of measurement equipment for microbe culturing in space - Contribution to the MOREBAC experiment,

part of the MIST-project

Oscar Årling

Teknisk- naturvetenskaplig fakultet UTH-enheten Besöksadress: Ångströmlaboratoriet Lägerhyddsvägen 1 Hus 4, Plan 0 Postadress: Box 536 751 21 Uppsala Telefon: 018 – 471 30 03 Telefax: 018 – 471 30 00 Hemsida: http://www.teknat.uu.se/student

Abstract

Construction, programming and testing ofmeasurement equipment for microbe culturing inspace Oscar Årling

Many different bacteria have essential roles in the process of recycling organic waste, making them useful tools when it comes to establishing artificial ecosystems, a key technology to master in the expansion of human space travel.

In order to further investigate bacteria growth conditions during space travel, the MOREBAC experiment was formulated. The objective was to design an experimental setup and develop measurement equipment with the capability of confirming successful resuscitation of freeze-dried bacteria in space by measuring bacteria growth, on-board the student-built MIST-satellite.

The experimental setup prototype consisted of an acrylic chip wherein the bacteria would be placed during experiments and an optical measurements configuration using a photosensor with the purpose of detecting bacteria cell growth. For experimental environment monitoring, a temperature sensor and a pressure sensor were calibrated.

An Arduino Nano microcontroller was programmed to control all electrical components during measurements. During the optical density measurements blue dyed water and E.coli bacteria in nutrition media were used as test samples.

Provided varying blue dye or bacteria cell concentrations, in the form of dilution series and growth-over-time-series, the equipment proved capable of producing measurements that indicate the optical density of the test sample.

Furthermore, a prototype experiment protocol simulating events that will occur in the final experiment design, was implemented and was able to produce real-time monitoring graphs of optical, temperature and pressure measurements, as well as documentation of all events and measurement data.

ISSN: 1401-5757, UPTEC F 16062Examinator: Tomas NybergÄmnesgranskare: Maria TenjeHandledare: Håkan Jönsson

Summary in Swedish

Ända sedan den första månlandningen har en av mänsklighetens stora utmaningaroch strävanden varit att erövra större delar av universum. Att kolonisera andrahimlakroppar är ingen lätt uppgift när dessa inte ens tillåter växter att grönskaeller har en atmosfär som tillåter oss att andas. Men det som inte �nns kan manalltid försöka skapa. Så, för att vi människor någonsin ska kunna kolonisera andraplaneter behöver vi kunna återskapa den omgivning som jorden erbjuder så passlikt att vi faktiskt kan överleva i den.

För att skapa en sådan omgivning krävs många olika komponenter för att systemetinte ska fallera. I ett fungerande ekosystem krävs bland annat organismer som kanhantera avfall genom nedbrytning som därigenom tillåter andra organsimer attåteranvända avfallet. En mycket viktig komponent i alla ekosystem är bakterier,just på grund av deras delaktighet i nedbrytningen.

Bakterier kommer i många olika format och i deras mångfald �nner man att dekan uträtta många olika utgifter. Medan vissa bakteriearter orsakar sjukdomar,�nns det andra som fullbordar viktiga kretslopp i naturen som exempelvis kväve-cykeln.

På KTH pågår projekt kallat MIST (MIniature STudent satellite) där tanken äratt konstruera en satellit vari sju olika experiment ska utföras under satellitensomloppsbana kring jorden. Ett av dessa experiment är MOREBAC, som går ut påundersöka odlandet av bakterier i rymden, närmare bestämt frystorkade bakterieroch hur väl man kan lyckas återuppliva dessa från deras dvala.

I detta examensarbete har målet varit att konstruera mätutrustning för MORE-BAC:s räkning. Mätutrustningen ska tjäna syftet att kunna identi�era huruvidakoncentrationen bakterier ökar efter återupplivningsförsöket som kommer att skefrån frystorkad form. Dessutom ska utrustningen kunna kunna mäta relevantaförhållanden i omgivningen, i detta fall temperatur och tryck.

Alla elektriska komponenter, såsom sensorer, resistorer och LED kopplades till enArduino Nano microcontroller, som programmerades via datorn och sedan kunde

3

man via datorn skicka uppgifter till microcontrollern som i sin tur styrde de elek-triska komponenterna så att de utförde dessa uppgifter.

För att kunna odla bakterierna måste vi ha någonting att odla dem i, någotförslutet som inte tillåter bakterierna att �yga omkring hursomhelst, vilket degärna gör när gravitationen är liten. Vi använde ett genomskinligt chip med en in-loppskanal där näringsämnen till bakterierna kunde pumpas in och en utloppskanalför att kunna pumpa ut gas och vätska.

Eftersom vi vill kunna bestämma bakteriekoncentration utnyttjade vi det faktumatt bakterier absorberar mer ljus ju högre koncentrationen är. På så sätt kundevi använda en ljuskänslig sensor till att bedömma hur stor andel ljus bakteriernaabsorberade när de belystes med en riktad ljusstråle från en LED. Exempelvis,om en liten mängd ljus skulle släppas igenom bakterierna betyder det att de ab-sorberar en stor andel av ljuset, vilket tyder på en hög koncentration av dem.Mätningar på olika bakteriekoncentrationer gav oss därmed en bra referens tillvilka mätvärden som motsvaras av en viss koncentration. Inledningsvis, för atttesta den principen och för att undvika tidskrävande bakterieodlingsförberedelser,testades mätutrustningen på olika spädningar av färgat vatten.

Temperatursensorn och trycksensorn testades för olika temperaturer respektivetrycknivåer och påvisade båda två att de var linjärt beroende av mätvärdena utanmärkbara avvikelser, vilket innebar att de kunde anses som tillförlitliga för refer-ensmätningar av dessa storheter på intervallen 25 ◦C till 70 ◦C, respektive -80 kPatill 30 kPa.

När alla komponenter hade testats separat testades de alla samtidigt enligt exper-imentprotokollet, vilket innefattar alla händelser som kommer ske när satelliten ärsatt i bruk och det är dags för experimentet att utföras. Under testutförandet avexperimentprotokollet skapades, samtidigt som mätningarna pågick, en visualiser-ing med tre grafer föreställande mätningar på absorbans, temperatur och tryck.Dessutom dokumenterades alla händelser i en �l på datorn.

Från resultatet av utförandet av experimentprotokollet kunde det konstateras atthändelserna dokumenterades som de skulle och att mätutrustningen kunde följatemperatur-, tryck- och absorbansförändringarna som skedde. Dock antydde ab-sorbansmätningarna ett temperaturberoende, vilket innebär att den valda ljussen-sorn omkalibreras eller bytas ut mot en ljussensor utan temperaturberoende.

I fortsättningsarbetet kommer det behövas försök till återupplivning av frystorkadebakterier, utredning och åtgärd för ljussensorns temperaturberoende samt paral-lella experimentutföranden, med varierande parametrar så som tidsperiod, djuppå chippets bakteriebrunn.

4

Contents

1 Introduction 7

1.1 Aim of Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.2 Bacteria in Space . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3 Life Support Systems and Pocket Earth Ecosystems . . . . . . . . . 81.4 Detecting Bacteria Growth . . . . . . . . . . . . . . . . . . . . . . . 91.5 Studies of Previous Work . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Background 13

2.1 The MIST satellite project . . . . . . . . . . . . . . . . . . . . . . . 132.2 MOREBAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.3 The Employer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

3 Theory 15

3.1 Experiment Limitations . . . . . . . . . . . . . . . . . . . . . . . . 153.2 Serial Communication . . . . . . . . . . . . . . . . . . . . . . . . . 163.3 Experimental Preparations and Procedure . . . . . . . . . . . . . . 163.4 Experiment Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4 Design, Construction and Testing 20

4.1 Electrical Components . . . . . . . . . . . . . . . . . . . . . . . . . 204.1.1 Microcontroller and Computer Softwares . . . . . . . . . . . 224.1.2 Optical Measurement Components . . . . . . . . . . . . . . 234.1.3 Temperature and Pressure Sensor Calibration . . . . . . . . 244.1.4 Choosing Resistances . . . . . . . . . . . . . . . . . . . . . . 25

4.2 Chip design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264.3 Optical Measurement Con�guration . . . . . . . . . . . . . . . . . . 284.4 Arduino and Processing programming . . . . . . . . . . . . . . . . . 294.5 Dye and Bacteria Measurements . . . . . . . . . . . . . . . . . . . . 304.6 Experiment Protocol Testing . . . . . . . . . . . . . . . . . . . . . . 31

5 Results 32

5

5.1 Resistance tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335.1.1 Analytical Resistances . . . . . . . . . . . . . . . . . . . . . 335.1.2 Typical Photoresistance Measurements . . . . . . . . . . . . 35

5.2 Dye and Bacteria measurements . . . . . . . . . . . . . . . . . . . . 365.3 Temperature and Pressure Calibration . . . . . . . . . . . . . . . . 395.4 Experiment Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . 40

5.4.1 Logged Events . . . . . . . . . . . . . . . . . . . . . . . . . . 415.4.2 Real Time Monitoring Graph . . . . . . . . . . . . . . . . . 42

6 Discussion 44

6.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466.2 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

6

Chapter 1

Introduction

In this section, the underlying circumstances that motivate the MOREBAC-experimentare presented, such as the usefulness of bacteria in life support systems and arti�-cial ecosystems. Also, the aim of the project, what approaches were taken to getthere, and design limitations are all discussed.

1.1 Aim of Project

The aim of this master thesis was to develop measuring equipment for the detectionof growth of resuscitated freeze-dried bacteria, on-board a student built satelliteorbiting Earth.

Initiating the MOREBAC project, this �rst phase of the project served to establisha basis for other students to continue working on to reach a �nal product that willbe able to be incorporated into the MIST-satellite.

1.2 Bacteria in Space

Bacteria can be useful in many appliances related to the recycling of biologicalwaste, which would be particularly crucial on space missions where the resourcesthat are brought along must last throughout the duration of the mission.

Bringing bacteria out into space requires rigorous con�nement to avoid any con-tamination on-board the vessel. Furthermore, the bacteria are living cells thatrequire nutrition and the right environment to thrive.

7

One suitable solution to the need of keeping bacteria alive during long space mis-sions, is to use freeze-dried bacteria that are resuscitated when the moment isright for the bacteria to be put into use. However, this requires a reliable resusci-tation method where enough bacteria survive and are able to reproduce. To verifythat the bacteria indeed survive the process of resuscitation, measuring equipmentcapable of detecting the bacteria growth is needed.

1.3 Life Support Systems and Pocket Earth Ecosys-

tems

Why do we need humans operating in space? Why can't we just have robotsperforming the work of humans? Robots are powered by electricity, and unlike usorganic humans, need no nutrition intake and do not have as strict environmentalrequirements. Furthermore, robots have no juristic rights and thus can be usedfor tasks that would put a human in harm's way.

However, there are many reasons why humans should be sent into space, and notjust robots. For one, sending humans into space helps improving our understandingof how the space environment a�ects the human body. Humans are also more aptat solving a larger variety of tasks as we can use our knowledge and problem-solving skills to �nd solutions to any problems that occur. So far, no arti�cialintelligence has been developed that can replace that ability. The machines areprimarily tools to save time, reduce danger, and increase precision.

When humans are sent out on space�ight missions there is no guarantee that theywill survive the trip, but there is however an enormous e�ort put into loweringthe risks of all forms of exposure to danger. There is always a plan to bring theastronauts back to Earth alive after the mission's completion.[1]

The �rst moon landing proved that humans can actually visit other celestial bodiesand return safely. Knowing this is possible, it is not an unthinkable notion taking itto the next step - staying for a longer period of time, and ultimately even colonizeother planets. But to do this, we would have to be able to create a pocket Earthecosystem: An enclosed volume with arti�cial atmosphere resembling the Earth's,where humans and other life forms can live without wearing space suits or carryingany life aid.

In order to create a ecosystem that sustains an environment in which humanscan survive, there are many conditions that must be ful�lled. For example, theair composition must be regulated so that the air remains breathable instead of

8

becoming choking or toxic. Other vital requirements are clean water, food withessential nutrients, livable temperature and pressure levels, and protection fromradiation.[2] Normally, all this is available beneath Earth's atmosphere but duringspace missions all that is available are the things that are brought along aboard,therefore a substitute for all the essentials Earth provides has to be presented. Thesystem put in place to enable humans to survive during space�ights, by regulatingthe environment and supplying life-necessities, is called a life support system.

Imitating the conditions found in Earth's atmosphere, a life support system of apocket Earth ecosystem would have to be very immaculately designed. One of themost important aspects of a sustainable pocket Earth ecosystem is the recyclingof biomaterial, and this is where bacterias play a substantial role.

In the process of biomaterial recycling all waste is decomposed, which can thenbe reused as nutrition for organic life forms such as plants and bacteria. However,all cycles need to be closed for the system to be able to be sustained withoutintroducing new material to the system. An ecosystem should, after a long periodof time, show no signs of having essential building blocks ending up amassed andunable to partake in the circulation.[3]

When organisms die, certain bacterias and fungi that exist in the soil decomposethe proteins into smaller elements. After several steps in the ecosystem's chain ofpossible processes, the elements �nd their way back to where they once startedwith the help of bacteria, other living organisms and also things like di�erentweather phenomena.

In the nitrogen cycle, a number of bacterias interact with nitrogen compounds indi�erent ways. Decomposing bacteria breaks down organic waste material intoammonia and ammonium ions, and nitrogen �xing bacteria �x nitrogen gas fromEarth's atmosphere into the soil. Denitrifying bacteria helps release nitrogen backinto the air. Together, these bacteria are essential in creating a balance of nitrogenin its varying compositions. [4]

1.4 Detecting Bacteria Growth

The main focus of this thesis was to develop a method of veri�cation for bacteriasurvival and reproducibility after the resuscitation from freeze-dried form.

To measure bacteria growth, suitable techniques had to be considered from pre-existing techniques. One technique which is prevalent in laboratories of biologicalresearch (by the use of spectrophotometers) is measuring the portion of light that

9

passes through a substance containing the bacteria. The substance's light trans-mission rate is referred to as optical density, a property that relates to the howmuch light the particular bacteria species absorbs, and its concentration, thusmaking this technique an excellent approach for detecting bacteria growth.

With access to the right nutrition and environment, a bacteria cell divides peri-odically into two. The new cell is identical and will also reproduce in the samefashion. This means that, as long as the growth conditions are right, a populationof bacteria cells will continuously duplicate at a certain time interval.

E.coli was used as the subject bacteria in this thesis work, preferred for its speedygrowth rate. Depending on the environment, the duplication rate of E.coli canvary immensely, from 20 minutes in optimal conditions to a much slower rate of12-24 hours which is its estimated intestinal tract duplication rate. [5]

The life-cycle of bacteria is usually divided into four phases that are characterizedby the bacteria cell concentration curve slopes, from the culturing of a batchperformed in a closed system.

Initially, during the lag phase, the bacteria prepare to reproduce and have not yetstarted multiplying noticeably. Then, during the exponential phase the bacteriagrowth skyrocket until the resources starts to thin out. Next, as the growth issuppressed in the stationary phase, the bacteria stop growing in numbers and stayat a steady level. Finally, in the death phase, the cell population declines as aresult of cell-deaths happening more often than cell-division, due to all the toxicmetabolic waste accumulated during the span of the bacteria cell culturing, as wellas the shortage of nutrition and oxygen. [6]

Even though the number of living bacteria cells decrease during the death phase,absorbance measurements of the culture will not yield lower value results as evendead cells contribute to a higher absorbance, meaning that the absorbance mea-surement will indicate the concentration of the sum of living and dead bacteriacells.

10

1.5 Studies of Previous Work

In preparation of the thesis work, similar studies that have been performed involv-ing monitoring of micro-organism growth were studied.

In the paper Growth monitoring of a photosynthetic micro-organism (Spirulinaplatensis) by pressure measurement, estimations of concentration levels of the pho-tosynthetic cyanobacteria Spirulina plantensis were made through pressure mea-surements.

Much like in the MOREBAC experiment, the bacteria were cultured inside a closedsystem. Since the photosynthesis of the cyanobacteria resulted in continual releaseof oxygen, the pressure levels were constantly increasing, requiring pressure regu-lation every now and then, when a certain pressure level was reached. Therefore,pressures changes had to be tracked cumulatively to account for the release ofpressure due to regulation.

The method of using pressure measurements to monitor the concentrations wasvalidated using physiological models that involved the cell metabolism and itsmaterial balance relations during growth and its e�ects on the pressure inside theclosed volume.[7]

This technique could be a good compliment to the absorbance measurements inthe MOREBAC experiment, since pressure measurements will be performed re-gardless, as it will be necessary for pressure regulation. However, determining thecorrelation between concentration and pressure will require models that describeall the physiological circumstances of the growth process well, which will depend onwhat bacteria that will ultimately be the subject for the experiment. For example,factors like pH might be necessary to know in order to calculate the concentration,as was the case for the Spirulina plantensis culturing measurements. This wouldrequire more measuring equipment, testing and research, which could be di�cultto �t in the frame of the MOREBAC experiment.

Another study, Design, Operation, and Modeling of a Membrane Photobioreactorto Study the Growth of the Cyanobacterium Arthrospira platensis in Space Condi-tions, compared di�erent methods of measuring micro-organism growth in spaceconditions. The methods tested were optical density, pressure and pH measure-ments. The experiment was carried out over 500 hours to what long-term e�ectsradiation had on measurements.

Like in the previously discussed study, pressure measurements were conducted todeduct bacterial growth. To make sure that the pressure sensor was measuringon oxygen, a hydrophobic membrane was used to separate the oxygen from the

11

liquid phase. Pressure increased as a consequence of oxygen evolution duringthe photosynthetic process, during which the pH increased as well due to carbonconsumption, providing yet another way to indirectly measure bacterial growth.However, to maintain cultivation, the pH-value had to be regulated when the levelsstarted to inhibit growth. Keeping the pH in the range of 8.3-10, carbon dioxide(CO2) was supplied to the culture to bring the pH-level back down.

In the context of the MOREBAC experiment, the pH-measurements would bedi�cult to perform since additional operations involved in regulating pH, as wellas putting a pH-probe in contact with the bacteria growth chamber would becomplicated, requiring additional channels in the chip.

When it comes to the long term e�ects of radiation on the equipment, the compo-nent used for optical density measurements, a photodiode, could only give reliablemeasurement data up to 300 hours into the experiment. In other words, the opticalmeasurements became inaccurate over time, possibly due to radiation exposure.However, pressure measurements did keep producing reliable measurements datathroughout the whole duration of the experiment, with accurate and precise re-sults. [8]

12

Chapter 2

Background

This section familiarizes the reader with the context in which the work has beencarried out.

2.1 The MIST satellite project

All over the world, researchers and university students design and build smallsatellites, called CubeSats, which are sent into orbit around earth once they areoperational and certi�ed by the space agency of the a�liated region. The MIST-satellite (Mini Student satellite) project is a cooperative e�ort among universitystudents to construct a satellite containing several scienti�c experiments, whichare to be executed inside the satellite once it is orbiting earth. One of these isMOREBAC, which will be the only experiment carrying organic material.

The projects have been suggested by KTH Royal Institute of Technology, IRF theSwedish Institute of Space Physics and the two companies NanoSpace AB andPiezomotor AB. Sven Grahn is the project leader. The work started the 28thof January 2015 and is estimated to be �nished in 2017, with a launch in 2018.[9]

There are seven separate projects which are all developed by student groups. How-ever, since all the projects will share the restrictions of space and power consump-tion, it is imperative to establish a good communication and coordination betweenthe projects for everything to be able to run smoothly after launch.

13

2.2 MOREBAC

MOREBAC (Micro�uidic Orbital Resuscitation of Bacteria) was proposed by theDivision of Proteomics and Nanobiotechnology, KTH. The MOREBAC experimentwill be carried out aboard the MIST-satellite after students have presented theircontributions to its development and all equipment is indeed operational.

The purpose of the experiment is to investigate the resuscitation process of freeze-dried bacteria in the satellite during its orbit around Earth.

MOREBAC will be carried out by master students from KI, KTH, SU and UU,working in succession to develop the experimental setup.

2.3 The Employer

This thesis was carried out at the Department of Proteomics and Nanobiotechnol-ogy at Science for Life Laboratory, SciLifeLab, under the supervision of researcherHåkan Jönsson, who is also a lecturer at KTH Royal Institute of Technology.

SciLifeLab, is a center of national resource for molecular biosciences with focuson health and environmental research. SciLifeLab is a collaboration between fouruniversities: Karolinska Institutet, KTH Royal Institute of Technology, StockholmUniversity and Uppsala University.

14

Chapter 3

Theory

This section further explains the scope of the project, aspects such as limitations,communication, and the experiment formulation.

3.1 Experiment Limitations

The space environment and the size of the satellite bring about limitations for allthe MIST-satellite projects. Each project has been allotted a partition of the totalspace inside the satellite. The alloted space for the MOREBAC experiment is ap-proximately 70x70x40 mm, but these dimensions are subject to change dependingon whether other experiments will need more space inside the satellite.[10]

Furthermore, because of the limitations on the power usage, the equipment needsto have low voltage levels and low power consumption to be able to hold up tothe shared capacity. A lower power consumption also means better temperaturecontrol since less heat will omit from the equipment during use. The maximumvoltage the components will be supplied with will be limited to 5 V. Anotherrestriction that will have to be taken into account is the materials. For example,some plastics are prone to out-gassing, which is a slow evaporation of the material,emitting gases to the nearby environment. This is mainly a problem when thematerial is exposed to vacuum.[11] Materials that are porous should be avoidedwhen it comes to making the culturing chip, since all gases, �uids and of course thebacteria themselves need to be contained and not leaked out. Furthermore, unless acovering layer that protects the equipment from radiation exposure is implemented,the components and materials must be able to withstand radiation.

15

3.2 Serial Communication

Microcontrollers are helpful tools that enable communication between computersoftware and electrical circuitry. Commands can be sent from the computerthrough a serial cable informing the microcontroller which of the connected com-ponents should be active and for how long. The information �ow goes in bothdirections also allowing the computer to access measurement data from the mi-crocontroller. The work in this thesis heavily depends on the microcontroller'scentral role in the testing of electrical components and the monitoring of the bac-teria growth experiments.

3.3 Experimental Preparations and Procedure

For some of the measurement equipment testing, a sample substance containingbacteria was required and therefore the sample had to be prepared before anymeasurement could be initiated.

The culturing of E.coli bacteria was conducted in a biology lab where many di�er-ent bacteria were handled on a daily basis. Consequently, the risk of contaminationhad to be taken into account when handling the bacteria samples, because if otherbacteria started growing in the sample, the measured absorbance would not bethat of the subject bacteria. So, when a sample container was exposed to the openair, a burner was used around its edges to mitigate the risk of contamination bykilling o� other bacteria that might have lingered in the air.

To start the culturing process, the original batch of E.coli were taken out fromthe freezer, and from the batch a small amount of bacteria cells were extractedusing a cell spreader, which is a long thin plastic tool that facilitates bacteriaextraction. The cell spreader with the attached bacteria was used for stirring in aplastic container containing nutrient media so that the bacteria would mix into themedia. Then the container was put into an incubator so that the bacteria couldthrive and multiply. The incubator was constantly shaking the bacteria samplewith a rotating motion, while keeping the temperature at a constant 37◦C.

After 8 hours in the incubator, the E.coli had grown so much that they visiblyclouded the media. Thus, the media with these bacteria had become a quali�edcandidate for the base sample for dilution and growth experiments.

Once the base concentration sample had been produced, it was time for either adilution series or a time series to be measured in the optical measurement device

16

prototype. For absorbance calibration of the dilution series, each dilution's op-tical density was also measured using a spectrophotometer. For the time series,the optical density was measured in the spectrophotometer just before startingand after �nishing the constructed measuring device measurements. The spec-trophotometer measurements were performed by putting the samples in cuvettes,small measuring jugs with square-shaped cross sections, which were put into thespectrophotometer.

Using a syringe and tubes, the sample that was going to be measured was injectedinto the chip through the inlet channel and the chip was placed in measurementposition, between the mechanical iris and the photosensor, with its surface perpen-dicular and centered to the light beam that would be emitted by the LED duringa measurement.

To be able to produce any measurements, the mini-USB had to be connected tothe Arduino Nano and the computer and then the Arduino script uploaded ontothe microcontroller.

Lastly, the measurement device was placed into a light-impenetrable carton boxand the Processing script was started. The monitoring process had begun andmeasurements were being stored on �le.

3.4 Experiment Protocol

Experiments that will be performed during the satellite's orbit must be thoroughlytested on earth beforehand since there is only once chance for each experiment tobe executed after the satellite's launch. Therefore a protocol for the experimentalprocedure needs to be designed and tested. Every event that takes place in prepa-ration of and during the experiment should be documented over the timespan ofthe experiment along with the measurement data.

Also, before and during the experiments, a number of variable checks and actionssuch as temperature and pressure control must be performed to ensure that theright experimental conditions are ful�lled. All these events need to be part of theprotocol.

At the �nal stage of the project, when the measuring equipment development is�nished it will be mounted onto the MIST-Satellite and be connected to the maincomputer from where all commands of the protocol will be sent, and to where allmeasurement data will be sent back.

During the bacteria growth detection experiment, many di�erent events occur.

17

Some are periodically reoccurring and some only happen once. For example, mea-surements of absorbance, temperature and pressure need to be performed through-out the whole experiment while events like injection of media into the bacteria chiponly takes place once.

The experiment protocol script handles all these events while storing importantinformation from the whole process, so by implementing it the experiment can berealized and more easily analyzed.

Here is a preliminary version of how the experiment protocol looks. Note howeverthat the only events that could be carried out were the actual measurements, andno mechanical or thermal events such as temperature and pressure regulation orin�ow of medium.

The events that take place during the experiment are described below.

Temperature control:

Monitor the temperature and regulate it, to ensure the right conditions for themedium to be �uid before injection into chip and for bacteria to grow.

- Temperature measurement at a close proximity of the chip's culturing chamber.

- Temperature regulation, if temperatures are out of acceptable range.

In�ow:

Injection of medium, containing nutritions and resuscitation liquid, through thechip inlet channel into the culturing chamber.

- Open chip inlet and outlet valves

- Injection of medium

- Close chip inlet and outlet valves

Pressure control:

Maintain a pressure level that is suitable for the bacteria to grow in and that doesnot strain the chip, as the pressure increases when gases are released in the celldivision process. Repeat following steps until pressure level is on that level.

- Measure pressure level at chip outlet

- If the pressure level is too high, open chip outlet valve for a short duration andthen close it

18

Absorbance measurement:

Monitor bacteria growth through absorbance measurements

- Measure optical density

Here follows the order in which events will take place during the experiment:

1. Temperature control, performed periodically throughout the experiment

2. In�ow, performed once to initiate resuscitation process

3. Pressure control, performed periodically throughout the experiment

4. Absorbance measurements, performed periodically throughout the ex-periment

Note that after the in�ow of medium, the pressure control and absorbance measure-ments are initiated and continue periodically with independent time steps.

19

Chapter 4

Design, Construction and Testing

In this section, the development and testing process is presented. This concerns thechoices of electrical components, the design of the bacteria chip, the con�gurationof the optical measurement device, the programming of microcontroller as well asthe execution of measurement tests.

4.1 Electrical Components

From a large online selection of electrical components, the appropriate sensors,LED:s and resistors were selected and ordered. All selected components adheredto the 5 V restriction of the experiment and can be seen in Figure 4.1.

20

Figure 4.1: Electrical components used to construct measuring device. a: Micro-controller Arduino Nano (Digikey, A000005), b: LED (Thorlabs, LED591E),c: Resistors (1.2 kΩ, 10 kΩ), d: Photosensor (Digikey, Parallax Inc. 350-00009), e: Temperature sensor, (Digikey, MCP9700A-E/TO), f: Pressuresensor (Digikey, MPXV5100DP)

The testing of the electrical components of the measuring equipment was doneon a wiring breadboard, a board with a grid of pinholes for connecting loose endwires and pin components. How all the components were connected can be seenin Figure 4.2.

21

Figure 4.2: Schematic of all electrical components used

In the measuring device prototype, the microcontroller was connected with theelectrical components in such a way that four di�erent outputs from the micro-controller could activate either the LED, the photosensor, the temperature sensoror the pressure sensor.

Also, connections were made from the photosensor, the temperature sensor and thepressure sensor to the microcontrollers input pins. These connections establishedmeasure points for the sensor measurements.

The LED was serially connected with a 1.2 kΩ resistor, which is called voltagedivision, preventing the voltage from overloading the LED. Similarly, the pho-tosensor was serially connected with a 10 kΩ resistor. This voltage division wasnecessary for creating a new measuring point, since a voltage drop depending onthe current of the circuit would occur over the serial resistance. Otherwise, with-out the resistance, we would just be measuring the output of the microcontrollerwhen measuring over the photosensor. Thus, with the new measuring point, thevoltage over the photosensor varied whenever its resistance or current varied. Thecon�guration can be seen in the schematic in Figure 4.2.

4.1.1 Microcontroller and Computer Softwares

An Arduino Nano, see Figure 4.1 .a, was used as microcontroller to control theother electrical components of the measurement device. It was mounted onto thebreadboard and connected with the components as can be seen in Figure 4.2.

22

The microcontroller has 32 pins, is capable of supplying an output voltage of 5V and its input resolution is 5/1024 V. Additionally, it has mini-USB socket forcomputer connection.

the Arduino software was used to program the behavior of the microcontroller. Inthe software, output voltage of the microcontroller's speci�ed pins can be activated,as well as input data from the pins connected to a circuits measure-points.

Processing, is a java-based software that provides an easy-accessible graphics userinterface that by default uses a drawing loop for visualization. It can also beused to send and receive serial information and thereby communicate with themicrocontroller. It was used during measurements, sending commands to activateelectrical components at speci�c times as well as storing and visualizing measure-ment data.

4.1.2 Optical Measurement Components

For the optical density measurements, apart from the serial resistors in the voltagedivisions, two electrical components were required: An LED, as seen in Figure4.1.b, to emit light, and a photosensor, as seen in Figure 4.1.d, to measure incominglight.

Additionally, the wavelength of the light emitted from the LED needed to beable to be detected by the photosensor as well as be absorbed by the subjectbacteria depending on its concentration. A commonly used wavelength for E.coliabsorbance measurements (and many other bacteria) is 600 nm, an orange-yellowcolor. Wavelengths within the visible light spectrum are also less harmful for theE.coli than for example UV-rays.[12].

Among the LED:s with a range of wavelengths close to 600 nm, an LED of 591nm was selected for the optical density measurement testing. The spectrum of theLED �tted into the intended bacteria's light absorption spectrum as well as thephotosensor's detection range.

To be able to perceive a small change in light level, which would imply bacteriagrowth in an absorbance measurement, the photosensor had to be sensitive at theinterval of change. Furthermore, since the microcontroller only has a resolutionof 5/1024 V, the range of the measured voltages needed to be as large as possiblefrom the lightest to the darkest light levels of the measurements in the experiment.Based on these criteria, the photoresistor seen in 4.1.d was chosen to be used asphotosensor for the measuring device. The photoresistor is characterized by havinga resistance that is light-dependent, going from a very high resistance at low light

23

levels to a much lower resistance at high light levels. That resistance changecorresponds to a voltage change over the component, which can be measured andused as an indicator for absorbance.

4.1.3 Temperature and Pressure Sensor Calibration

Some bacteria are known to not only withstand high temperatures, but even pre-ferring them. Extremophiles, as they are called, have optimal life conditions intemperatures where humans could not live. For example, Thermus Thermophiluswhich has a 68 ◦C optimum.[13].

E.coli however, along with the majority of bacteria species, prefer milder temper-atures. E.coli thrives in the human intestines where its optimum temperature of37 ◦C is found. The maximum temperature at which E.coli keep growing dependson what bacteria strain the particular belongs to. Generally the growth is inhib-ited at 41 ◦C. [14] E.coli can survive freezing temperatures, and can even growat low temperatures, although at a very slow rate, like 7 ◦C. [15] However, inthe MOREBAC experiment, the aim is to maintain the temperature around itsoptimum growth rate temperature.

To maintain temperatures and pressure levels within the right intervals, the sensorswith the purpose of measuring those quantities had to be calibrated, in addition tothe regulators acting to keep the levels in check. However, no testing of regulatorswas performed at this early stage of the MOREBAC project.

The calibration of the temperature sensor which stated range is −40◦C ∼ 125◦C was performed by putting the sensor in a block heater (SBH130, accuracy ± 1◦C) that provided temperatures from 25◦C to 70 ◦C. [16] All voltages measured bythe temperature sensor could be plotted to the corresponding temperature, thususing calibrated values from these measurements can later produce temperaturevalues from measured voltages in the environment of choice, such as in the MISTCubeSat.

The pressure sensor has two ports onto which tubes can be connected and a voltageis measured that relates to the pressure di�erence. Similar to the temperaturecalibration, the pressure calibration was performed by having a pressure giver(PC20•25) providing pressure levels ranging from -80 kPa to 30 kPa, measuredwith a ± 2.5 % accuracy.[17] This range allows the pressure to be measured for�uctuations in one direction up to 80 % of the atmospheric pressure, 101 kPa,which will be the operating pressure inside the chip during the experiment.

24

4.1.4 Choosing Resistances

Besides the innate resolution limitation of the microcontroller, by the 1024 mea-suring points available, there are �ve major factors that a�ect the resolution of anoptical measurement. These factors are the photosensor, its serial resistance, theLED, the chip thickness and the absorbance of the test sample at its maximum.Considering that the maximum absorbance of the test sample will likely have atypical value at the bacteria cell death phase, and that the chip characteristicscannot be varied easily without having to mass produce all the alternative chipmodels, a reasonable way of �ne-tuning the resolution was to decide on an LEDand photosensor and then test for varying resistances.

Depending on the maximum light exposure of the photosensor, the resistance con-nected in serial had to be chosen with consideration to that. In the experiment,as it would be performed aboard the satellite, the typical light level at maximumexposure would correspond to the case where the chip and freeze-dried bacteriaare placed between the LED and the photosensor at the stage of the experimentwhere the resuscitation process has not yet begun.

However, instead of calculating the typical maximum light level, the typical resis-tance of the photoresistor at the maximum light exposure could be used since theresistance of the photoresistor changes with exposure to light. Having a voltagedivision over the two components in serial, the resistance of the photosensor, Rp,could be calculated by:

Rp =R

VsVp

−1

where R is the serial resistance, Vs the supply voltage and Vp the voltage over thephotosensor.

By varying R and measuring Vp, and seeing that the photoresistor's resistanceindeed did not change remarkably the measurements indicated a typical resistancearound 10 kΩ.

After having calculated the resistance of the photosensor at maximum light expo-sure from the LED, the serial resistor could then also be determined.

To get a better understanding of how the choice of the serial resistance a�ectedthe outcome of the measurements, simulations were made over the resistance spanof the photosensor. Also, taking into account what serial resistance would presentthe highest sensitivity to a change of light level, the derivative of the voltage overthe photosensor with respect to the photoresistance was derived. It was foundthat the derivative was the largest at R = Rp, which meant the sensitivity was thehighest for that choice of serial resistance.

25

Formula for the sensitivity of the voltage with respect to a change in photoresis-tance:dVpdRp

= VtotRp

(R+Rp)2

A graph of the derivative was plotted for di�erent choices of R showing at whatresistances the optical measurement device would be most sensitive to a change inlight, see 5.1, page 33.

The serial resistance 10 kΩ was decided upon because it was close to the typicalmeasured photoresistance and required no more than one resistor from the resistorsavailable.

4.2 Chip design

A prototype chip in which the bacteria can be placed and start growing after theresuscitation needed to be designed. In order to keep bacteria in place a culturingchamber was designed to be a cylindrical cavity inside the chip, with the axisperpendicular to the chip's top and bottom surface.

Additionally, the chamber had to have an inlet for in�ow of resuscitation mediumand an outlet for pressure balancing when media is injected through the inletchannel and for the bacteria growth phase, which will otherwise result in elevatedpressure levels, risking damage on the chip.

The inlet and outlet channels were made very narrow, 0.2 mm, to increase thein�uence of the shear stress exerted upon ingoing and outgoing �uids and thusimproving the control of the �ow. These channels are connected with the chamber,continue outwards in a direction parallel with the long side of the chip and halfwayto the edge they make sharp right-angled turns towards the surface of the chip withthe largest area, corresponding to the bottom of Figure 4.3, until they reach thevery surface.

For optical density measurements it was important for the surfaces of the chip,especially at the bacteria chamber, to be as plane and smooth as possible in orderto disperse a minimal amount of the light that is going through the chip.

The �rst chip prototype was produced at KTH Machine Design prototyping center.The model used for this was created in the CAD-program AutoCAD, where a 3D-model was constructed. From the model, 2D-images were extracted from di�erentangles to be used as reference for creating the chip prototype. The chip wasdivided into layers since the machine drill only permitted drilling from a surface and

26

inwards with a drilling area that could not become larger as the depth increased.Therefore, creating a hole inside a solid block for example would not be possiblewith the drilling technique available. Had 3D-printing been an option however,which it wasn't because of the budget, there would not have been a need for asmany layers, but the fact remains that at least two layers are required to enablebacteria to be placed properly into the bacteria chamber.

Screw-holes were required to go through each layer of the chip so that the layerscould be assembled by being screwed tightly together.

Transparency of the material is absolutely necessary for the optical density mea-surements. Furthermore the chip needs to withstand varying temperatures andpressures. Acrylic was chosen for the �rst prototype since it was a transparentand hard material that could be shaped according to the 3D-model design, andwas available at KTH Machine Design prototyping center. In future work the chipshould be tested for the range of temperatures that it will be exposed to during theexperiment aboard the satellite, but to start with testing and further developingof the chip design was in focus.

Figure 4.3: A 3D-model of the �rst chip prototype, with screw-holes in each corner,a chamber in the center and an inlet channel as well as an outlet channel goingfrom the chamber.

27

Table 4.1: Chip DimensionsChip length 76.2 mmChip width 25.4 mmChip thickness 2 + 3 + 2 mmChamber diameter 10 mmcrew-hole diameter 5 mmInlet diameter 0.5 mmChannel width 0.2 mm

In table 4.1, the dimensions of the chip are speci�ed. The Chip thickness was laterdivided into three layers instead of two in order to create a smoother surface atthe chamber area. The top and bottom layer were each 2 mm and the middle layer3 mm.

4.3 Optical Measurement Con�guration

It was important to create consistency in the optical measurements in order toattain an acceptable accuracy. The main problems were to focus the LED so thatit was directed straight towards the photosensor, to keep the bacteria chip in alocked position where the bacteria would be centered, and also, to have the sameambient level of light at the photosensor for each measurement.

In order to establish a steadfast con�guration where the LED and the photosensorcould be locked in place aligned with each other, parts made for optical experimentswere used. Included in the optical setup were two steel beams used for alignment,three block holders enabling mounting of the LED and the photosensor as well asimproving the balance, and one mechanical iris for creating a small hole throughwhich the light could pass through and be focused on the bacteria chip chamber.See Figure 4.4. Furthermore, during measurements, the whole con�guration wasenclosed by a carton box to ensure an ambiance as dark as possible.

28

Figure 4.4: Optical measurement con�guration. The black parts, from left to right:Block holder with the mounted LED, another block holder, the mechanical iris,and then a block holder with the mounted photosensor. The chip was placed in aslit made in a piece of cellular plastic that enabled the chip chamber to stand in asteady position in the center of the light beam.

4.4 Arduino and Processing programming

The Arduino Nano microcontroller was programmed in the Arduino software. AnArduino script was written that included the responses of the microcontroller toserial information that would be sent from the Processing script on the com-puter.

In the Arduino script, all the output pins of the microcontroller were assignednumbers corresponding to what component was connected to them. These pinswere used for supply voltages during measurements, keeping components active orinactive at the right times. Similarly, the microcontroller's input pins were assignednumbers corresponding to what measuring point in the circuit that was connected.Through these input pins the microcontroller collected measurement data whichwas sent back to be stored on the computer by the Processing script.

Before the experiment protocol was implemented, every measurement that needed

29

to be made required the Processing script to be run once. In such a measurement,Processing would send a serial message with information about what action to beperformed, the microcontroller would receive the message and perform the speci�edaction and send the data back to Processing which would save the data to a �leon the computer.

4.5 Dye and Bacteria Measurements

Since bacteria must be cultured before use, and also need delicate handling, usingblue dye as a substitute sped up the initial testing phase. But once it had beencon�rmed that the measuring device could detect a di�erence in light level, bacteriahad to be tested as well.

In the case of the dye experiments, a base concentration of 6 drops of blue colormixed into 100 ml water was diluted down until there was just a hinge of blue visi-ble, each time decreasing the concentration down to a half of the previous sample.For the di�erent concentrations, the optical densities were measured in a spec-trophotometer to be compared with the prototype measurement equipment.

Bacteria dilutions were performed in the exact same way, except that the basesample consisted of E.coli bacteria that had been cultured overnight in nutrientmedia. Furthermore, the dilution of the bacteria sample were carried out untilthe spectrophotometer showed a OD-value (optical density) below 0.1, providinga dilution series ranging from OD = 0 to the optical density of the base sam-ple. The spectrophotometer was calibrated to a sample containing only media,corresponding to OD = 0.

In order to determine whether the equipment could indeed detect the increase ofbacteria after some time in the incubator, a time series experiment was carriedout. This means that absorbance measurements were made periodically on thesample during its growing phase in the incubator. A sample was cultured overnightin an incubator and before injecting the sample into the bacteria chip it wasdiluted with more medium down to a tenth of the previous bacteria concentration,to make sure that it was supplied with enough nutrients and started on a lowconcentration.

30

4.6 Experiment Protocol Testing

An experiment protocol script was written in both the Arduino and Processing soft-ware, as the Arduino Nano needed to be programmed for the speci�c commandsthat it would receive from the computer. A bacteria growth experiment was per-formed and monitored according to the experiment protocol. In other words, allevents were logged and a graph was produced, showing all measurements data. Thebox containing the measuring device was placed into the incubator and measure-ments were started immediately without letting the box air reach the equilibriumtemperature. This was done to be able to see how the temperature sensor behavedas the temperatures slowly increased to the that of the incubator. Also, it enabledus to see whether the other measurements were a�ected by the temperature level.After about 3.5 hours the box was taken out into room temperature for an hourbefore stopping the measurements, providing measurements from all the sensorsduring a decrease in temperature.

31

Chapter 5

Results

In this section, results from the developing process are presented as well as resultsof measurements performed after the design choices had been implemented.

When the chip had been manufactured, all components had been assembled, themicrocontroller connected to the optical measurement device and the environ-ment control sensors, the prototype measuring equipment was ready to performmeasurements. In order to identify the parts of the measuring device see Figure5.1.

32

Figure 5.1: Assembled measuring device prototype. A: Pressure sensor with con-necting tube. B: Temperature sensor. C: Microcontroller. D: Bacteria culturingchip. E: Optical measurement device.

5.1 Resistance tests

Both analytical simulations and experimental measurements produced informationon what behavior could be expected from di�erent choices of resistances.

5.1.1 Analytical Resistances

The graphs made from the analytical formulas show how the choice of serial resis-tance can a�ect the outcome of the optical measurements, for the whole resistancespan of the photoresistor. Over this resistance span and with a supply voltage of5 V, the graph in Figure 5.2 shows what measured voltages can be expected whenchoosing one of the three represented serial resistances of varying magnitudes, andthe graph in Figure 5.3 show how the sensitivity to a change of photoresistancea�ects the measured voltages for the candidate serial resistances.

33

Figure 5.2: Simulated results for threeresistances of varying order of mag-nitudes for the resistance span of thephotoresistor.

Figure 5.3: Change in photoresistancefor two candidate values of serial re-sistance, R1, for the resistance span ofthe photoresistor.

In Figure 5.2, it can be seen that for a large serial resistance a substantial pho-toresistance change is necessary to cover the voltage measurement span, meaningsmall �uctuations in light level are harder to detect. Conversely, for smaller serialresistances choices, a large voltage span is covered by a small change in photore-sistance at the highest light levels. For that photoresistance interval a �uctuationof light is more easily detectable. Hence, in comparison R = 10 kΩ o�ers a betterresolution than R = 200 kΩ, but is not as linear. However, a linear relation is notnecessary since photosensor calibrations will be able to provide a good mappingfrom measured voltages to optical density, when all components of the experimenthave been more precisely de�ned.

In Figure 5.3, it shows for both the serial resistances that the highest sensitivity toa change in photoresistance can be found where the photoresistance and the serialresistance are the same, since the voltage derivative peaks at those points. Thiscon�rms that the resistance can be chosen to give a high sensitivity to a smallerinterval by choosing one that is of the same order of magnitude as the typicalphotoresistance.

34

5.1.2 Typical Photoresistance Measurements

Produced from measurements on the photoresistor for a wide range of serial re-sistances, the following results show the typical resistance of the photoresistor atmaximum light exposure during the optical density measurement.

Figure 5.4: Typical resistance of the photoresistor at maximum light exposure,from testing resistances in the interval 10 - 500 kΩ.

As shown in Figure 5.4 the typical photoresistance at maximum light exposurecan be expected to be found inside the interval 9.8 kΩ and 11.2 kΩ. In order tominimize the number of resistance components and still remain within this interval,it was decided that a serial resistance of 10 kΩ would be used.

35

5.2 Dye and Bacteria measurements

The dye and bacteria measurements graphs show how well the equipment, afterthe choice of resistances, can detect a di�erence in optical density. The resultsfrom the dilution and time series measurements show that the equipment doesin fact manage to produce measurements that imply a clear relation between themeasured value and the optical densities of the test samples, on the whole spanof opacities. In Figure 5.5, the graph shows that the optical density measure-ment equipment can distinguish a di�erence in absorbance when subject to bluedye dilutions. The same applies to the bacteria dilution measurements that wereperformed, showing a similar curve, as can be seen in Figure 5.6. In both thesecases, the sample substances were contained in 10 mm thick cuvettes, small-sizedmeasuring jugs with square-shaped cross sections. Even if the optical path lengthin these measurements was longer than in the �nal prototype, the results wouldat an early stage reveal whether the optical sensor could in fact detect the varyingconcentrations of the samples.

Figure 5.5: Blue dye dilution measure-ments with serial resistance of 10 kΩ.

Figure 5.6: Bacteria dilution measure-ments with serial resistance of 10 kΩ.

Spectrophotometer measurements on E.coli inside the thin chip gave an indicationof how well measured voltages correlated to the optical density. This also gaveaccess to optical density calibration data for the thin chip's optical path length inroom temperature.

Table 5.1 shows the results from the measuring device and spectrophotometermeasurements of the bacteria dilution test samples. These results can also be seenin the graph in Figure 5.7, showing how the measured voltages are correlated withthe optical densities.

36

Table 5.1: E.coli Dilution Measurements Inside Thin ChipDilution Rate 1 1/2 1/4 1/8 1/16 1/32 0Spectrophotometer OD 2.8 1.33 0.75 0.4 0.18 0.1 0Measured Voltages 2.61 V 2.42 V 2.32 V 2.26 V 2.24 V 2.22 V 2.21 V

Figure 5.7: E.coli dilution measurements: Comparison of optical density mea-sured in a spectrophotometer and voltage measurements made in the prototypemeasuring device.

Since the bacteria growth measurements were performed with the slightly di�erentsetup, using a glass chip of 2 mm thickness instead of using the 10 mm thickcuvette, the voltage span became much smaller because of the smaller optical pathlength decreasing the sample's in�uence on the light. This can also be seen in thebacteria growth time series measurement in Figure 5.8, where the curve showsa steady increase in voltage over time, which, in accordance with the dilutionmeasurements, translates to a bacteria cell concentration increase over time.

37

Figure 5.8: Measurements on E.coli growth inside thin chip.

Despite showing signs of growth, the growth rate was expected to be higher becauseof the E.coli's duplication rate of around 20 min. However, since that rate appliesto optimal conditions, and considering that the media temperature was room-temperated at the start of the growth process, the growth rate could not havebeen as high as at the optimal temperature. Furthermore, since the oxygen supplywas scarce, the bacterial growth was inhibited further.

38

5.3 Temperature and Pressure Calibration

Since both the temperature and pressure sensors were tested for two di�erentsupply voltages, 3 V and 5 V, the results could be compared to see if the lowersupply voltage could be a viable alternative in case the power consumption needsto be cut down.

Figure 5.9: Voltage measurementsby temperature sensor for temper-atures 25 ◦C to 70 ◦C at supplyvoltage 5 V.

Figure 5.10: Voltage measurementsby temperature sensor for tempera-tures 25 ◦C to 70 ◦C at supply volt-age 3 V.

A side to side comparison of voltages measured by the temperature sensor forvarying temperatures can be seen in Figure 5.9 and 5.10. The measurements showa linear trend over the interval, making the data reliable to use as reference inother temperature measurements on the same interval.

39

Figure 5.11: Voltage measurementsby pressure sensor for pressure dif-ferences -80 kPa to 30 kPa at supplyvoltage 5 V.

Figure 5.12: Voltage measurementsby pressure sensor for pressure dif-ferences -80 kPa to 30 kPa at supplyvoltage 3 V.

A side to side comparison of voltages measured by the pressure sensor for varyingpressures can be seen in Figure 5.11 and 5.12. The measurements show a lineartrend for the negative pressure di�erences, making that data reliable to use asreference in other pressure di�erence measurements for that interval.

5.4 Experiment Protocol

During the bacteria growth phase, every occurring event is logged according tothe experiment protocol that is executed. Additionally, the monitoring graphgenerator that was made is updated every time a measurement occurs, showing alltemperature, pressure and optical density measurements made since the startingpoint of the experiment.

An optimal time interval between measurements could not be determined since thetimings will be formulated from the temperature and pressure regulation system.Readings that are a few minutes apart are enough to enable monitoring of the pro-cess in an environment where temperature and pressure do not vary dramaticallyand do not need to be regulated often.

40

5.4.1 Logged Events

The experiment protocol script was able to generate a �le of logged events with ac-companying timestamps and, in the cases of measurement, measured values.

In the example of an experiment run, Figure 5.13, temperature and pressure checksalways yields the result that the levels are �ne and need no regulation. This isbecause no regulation of temperature or pressure had been implemented at thisstage. Nevertheless, these messages serve the purpose of showing how a typicalexperiment log �le could look like.

Figure 5.13: Real time monitoring log of events from temperature, pressure andoptical density measurements during bacteria growth experiment.

41

5.4.2 Real Time Monitoring Graph

The experiment protocol script includes a visualization tool that plots all mea-surements made in the experiment, Figure 5.14. The optical density measurement(represented as absorbance) is displayed in the lower graph in a green color scheme,the temperature as red in the middle graph, and lastly, the pressure as blue in thetop graph.

As can be seen in all of the graphs, a sudden change of behavior occurs around210 minutes in. This was caused by the change of environment due to moving themeasuring equipment box out from the incubator and into the room.

However, by looking at this result an unexpected discovery was made: The ab-sorbance measurement values decreased as the temperature decreased, indicating acorrelation between the light sensor's output and temperature levels. This temper-ature dependence could depend on either the LED, the resistors or the photosensor,or all of these components. The temperature interval was realistic as it started atroom temperature and rose to the incubator temperature.

For each quantity measured, a list of the current, average, lowest and highestvalues were listed in the information box to the left.

In the graphs, it can be seen that both temperature and optical density measure-ments show an increase over time until the change of environment occurred. Theslow temperature rise was due to the fact that the box with measuring equipmenthad to be closed for the sake of not letting any outside light a�ect the light mea-surements, leading to a slow heat exchange between room temperated air insidethe box and the heated air in the incubator.

However, the colder room-temperated air from the outside seeping into the boxwas enough to create a more turbulent air�ow inside the box, making pressure�uctuations that the pressure sensor measured.

42

Figure 5.14: Real time monitoring graph of temperature, pressure and opticaldensity measurements during bacteria growth experiment.

43

Chapter 6

Discussion

Regarding the data of temperature and pressure calibration measurements, the re-sults show a consistent behavior, making those sensors reliable for measurements inupcoming experiments, where the calibration data trend can be used for referencelevels.

Even though the temperature calibration measurements deviated somewhat fromthe mean value of the linear trend that is otherwise present, the results are su�-ciently consistent to be used as calibration data for test experiments. At least, untila temperature regulation system that is dependent on the measured temperatureswill be implemented.

The temperature sensor is unable to be placed inside the chip so it must be placedimmediately outside of it during measurements inside the satellite. The temper-ature inside the chip will reach an equilibrium level near that of the outside airmaking this a su�cient method of deciding temperature level, or probably evenbetter, as the outside air temperature can be regulated faster if the sensor is placedthere and can pick up �uctuations easier.

The pressure calibration measurements proved to also follow a linear trend, and avery consistent one at that, during all measurements where the sensor was subjectto negative pressure.

In a study where growth of the micro-organism Spirulina Platensis was monitoredby pressure measurements in a closed photobioreactor, the pressure was not regu-lated until reaching 130% of the atmospheric pressure, needing only to detect a 30kPa di�erence in pressure while giving precise results. This limit also had a marginto the maximum recommended pressure level of 150% of the atmospheric pressure.[7] In relation to this, the ±80 kPa pressure di�erence range available from the

44

selected pressure sensor should be more than su�cient especially if measurementswill show that the pressure does not increase very rapidly during the E.coli growthphase inside the chip. From the experiment protocol testing monitoring graph thepressure level did not show signs of increasing rapidly, or increasing at all. How-ever, if other bacteria should be selected for tests, such as cyanobacteria, pressurewill most certainly increase during growth.

Both the temperature and the pressure measurements show that the respectivesensors will give equally valid results whether the supply voltage is 5 V or 3 V.However, this does not guarantee that any of the components will sustain damagethat might be caused by operating at another voltage than recommended. Thiscould be important to consider before a long experiment is initiated.

The most unexpected result appeared when all the components had been assembledand the �nal testing was performed. Interpreting the results from the experimentalprotocol led to the realization that the absorbance graph from the bacteria growthmeasurements was misleading, due to the fact that, not only did the absorbancecurve follow the temperature curve closely in the beginning, but it also actuallyfollowed the temperature decrease at the moment which the measuring equipmentwas put into a room temperature environment. This is a clear indicator that acomponent involved in the absorbance measurements, either the photosensor, theLED or the resistors has a considerable temperature dependency. The decreasein absorbance should not occur since the concentration of bacteria cells (dead oralive) should not decrease even when the culture stops growing and starts dyingo�.

The dependency had previously not caused any problems, at least not in the dilu-tion measurements as temperatures had been constant. However, in the end whentemperatures did �uctuate and measurements of temperature and absorbance wereproduced simultaneously and comparable side to side, the issue was revealed.

This means that in the continued work, actions need to be performed in order towork around the temperature dependencies of the components, either by replacingthe components that are temperature dependent, with ones without dependencies,or by calibrating the absorbance measurements after temperature measurements,in an e�ort to diminish the e�ects of the temperature dependencies as much aspossible.

Calibrating to temperature data will require testing on each of all the involvedcomponents to see how they individually are a�ected by temperature. If thiscan work out successfully and absorbance can be measured despite temperature�uctuations, there will possibly be no need to replace any components.

45

During the growth and dilution measurements it was noted that the thinner chipyielded a voltage span that was considerably lower than measurements made on thecuvette which was a lot thicker. A thicker chip depth yields more precise resultsthan a thinner chip in terms of measurement resolution, since the optical pathlength becomes larger. This is equivalent to making the opacity di�erence muchlarger in high versus low bacteria cell concentrations for a thicker chip. However,one of the limitations of the experiment is the amount of alloted space it will haveaboard the MIST-satellite, and thus the chip thickness will have to be adjustedaccordingly.

6.1 Conclusion

Being one of experiments that will take place in the MIST-satellite, the MORE-BAC experiment was designed to further investigate bacteria culturing on space�ights, with focus on resuscitation of freeze-dried bacteria. In this master thesis,the development of the MOREBAC measuring equipment for detecting bacteriagrowth and monitoring of the experiment environment was conducted.

The approach for detecting bacteria growth was measuring absorbance, i.e measur-ing the rate of light passing through a sample substance containing the bacteria.Environmental control consisted of temperature and pressure measurements.

For the measuring equipment, sensors, an LED, resistors, wires and a microcon-troller were connected on a breadboard. Furthermore, for the absorbance mea-surements experimental setup, the con�guration consisted of the LED and thephotoresistor directed toward each other, kept in place with holders and steelbeams for stabilization and a mechanical iris for centralizing the light beam.

Being placed in the light beam between the LED and the photoresistor, a transpar-ent bacteria culturing chip containing the test sample was used during measure-ments. Also, a custom-made culturing chip prototype made of acrylic was designedand manufactured to better �t the environment of the MOREBAC experiment andthe way it will be executed.

The microcontroller was programmed to keep speci�c electrical components activeduring measurements, depending on the commands it received from computersoftware through serial communication. Processing was the computer softwareused for sending commands to the microcontroller, and the Arduino software wasused to program the microcontroller's responses to those commands.

Tests were performed to evaluate the electrical components' behavior. The pho-

46

toresistor used for the absorbance measurements was able to di�erentiate betweenvarying bacteria cell concentrations, making it appropriate for detecting bacteriagrowth. However, when temperatures were varied to see how the components be-haved, a temperature dependency was discovered, the optical measurement compo-nents could only be concluded to yield reliable data for experiments with constanttemperature.

Even if the aim is to keep the temperature at an optimal one for bacterial growth,it will likely �uctuate since the temperature regulation system will not be updatingmeasurements at every second, but rather at larger discrete time steps, and willonly kick in when reaching designated levels.

The temperature and pressure sensors proved able to measure temperatures andpressures following a linear trend. The power supply of the microcontroller of 5 Vwas used throughout most experiments but the temperature and pressure sensorswere also tested and worked just as �ne for the lower supply voltage of 3 V.

After having tested the sensors separately, an attempt at simulating the exper-iment as it will be performed aboard the MIST-satellite was made, except onlymonitoring absorbance, temperature and pressure measurements during bacterialgrowth. The experiment protocol was thus implemented, successfully carryingout measurements while simultaneously producing monitoring visualizations anddocumenting events.

6.2 Future work

For the MOREBAC experiment to be executed, there are still plenty of featuressurrounding the measuring equipment that need to be implemented. An imme-diate task to accomplish for absorbance measurements to become accurate is toinvestigate and handle the temperature dependency of the optical measurementcomponents. Performing separate calibration with respect to temperature on theinvolved components could be one way of still using the same setup without chang-ing any components. Otherwise, the components can be substituted for less tem-perature dependent ones.

Moreover, a life support system that can maintain the right environment for thebacteria to grow in needs to be designed. This should include pressure and tem-perature regulation, as well as a system that takes care of nutrition supply. Forthe pressure regulation, tubes with pinch valves that open and close to even outpressure when the pressure levels are too high, is one solution that has been con-sidered and should be implemented. In a meeting with the MIST-group at KTH,

47

it was suggested that the temperature regulation could be assisted by one of theother project-groups in charge of a neighboring experiment aboard the MIST-satellite.

One of the important challenges in the future work of the project will be to managethe resuscitation of freeze-dried bacteria inside the culturing chip. Before resusci-tation, if the freeze-dried bacteria require storage at cold temperatures during thewait, the temperature sensor must be calibrated for those temperatures. It mustbe researched what bacteria media should be used as resuscitation activator liquidand how it should be contained before injection into the chip.

Since mainly just the design of the chip has been performed, its performance itterms of heat and high pressure exposure and containment of content for longerperiods of time must be tested.

A factor to keep in mind when further developing the culturing chip, is the microgravity environment in the satellite. Since the gravity pull will be quite weak, the�uids and gases are prone to moving about a lot more. This induces obstacleswhen it comes to keeping the �uids and gases in the experiment in place. Onesuggestion of a solution to keep �uids and gases where they belong in the chipis implementing hydrophobic and hydrophilic �lters, used for keeping �uids andgases separate. A hydrophobic �lter can be placed in the outlet channel of the chip,obstructing liquids from passing further than the culturing chamber. Similarly, ahydrophilic �lter can be placed in the inlet channel, letting liquids pass on to theculturing chamber and preventing gases from going into the inlet channel. Nylonmembranes can be placed next to the �lters to hold them in place and protectthem from wear. [18]

Eventually, when the experiment will be carried out aboard the MIST-satellite inits orbit around Earth, the execution of the experiment protocol will be requiredto work �awlessly, requiring a functioning communication between the microcon-troller and the main computer aboard the satellite. Furthermore, the experimentwill consist of many parallel measurements where some are performed on a smallscale time frame as in over the course of a few hours and others on a longer timeinterval as in, for example, over the course of weeks.

As discoveries are made and the experiment becomes more well-de�ned duringthe testing and development phase, the more important communication with theMIST team becomes. More information on exactly what they can provide in termsof alloted space, power, help with temperature regulation and general informationabout the launch of the satellite should be requested, as well as requirements fromthem what they expect in terms of measurement frequency, measurement datasizes and allowed components.

Bibliography

[1] P. Plait, �Safe return to earth.� (2014-06-08). Retrieved fromhttp://www.slate.com/blogs/bad_astronomy/2014/06/08/back_to_earth_how_astronauts_get_back_from_the_space_station.html.

[2] J. Fernando, �5 massive problems we'd face living on another planet.� (2015-12-10). Retrieved from http://www.cracked.com/article_23222_5-reasons-living-other-planets-would-be-nightmare.html.

[3] Globalchange, �The ecosystem and how it relatesto sustainability.� (2016-10-24). Retrieved fromhttp://www.globalchange.umich.edu/globalchange1/current/lectures/kling/ecosystem/ecosystem.html.

[4] M. Society, �Nitrogen cycle.� (2016). Retrieved fromhttp://www.microbiologyonline.org.uk/about-microbiology/microbes-and-the-outdoors/nitrogen-cycle.

[5] K. Todar, �The growth of bacterial populations.� (2012). Retrieved fromhttp://textbookofbacteriology.net/growth_3.htmll.

[6] S. aid, �Bacteria.� (2012-05-09). Retrieved fromhttp://www.scienceaid.co.uk/biology/micro/bacteria.html.

[7] B. L. . 1309�1314, �Growth monitoring of a photosynthetic micro-organism(spirulina platensis) by pressure measurement,� tech. rep., Kluwer AcademicPublishers, (2001).

[8] G. C, J.-F. Cornet, and J.-B. Gros, �Design, operation, and modeling ofa membrane photobioreactor to study the growth of the cyanobacteriumarthrospira platensis in space conditions,� tech. rep., Laboratoire de GenieChimique et Biochimique, Universite Blaise Pascal, (2005-02-25).

[9] A. Gårdebäck, �Studentsatelliten mist.� (2016-01-13). Retrieved fromhttps://www.kth.se/sci/centra/rymdcenter/studentsatellit/studentsatelliten-mist-1.481707.

[10] (2016-11-28). Information from supervisor Håkan Jönsson.

49

[11] J. Coonrod, �What is outgassing and when does it matter?.� (2010-11-19). Retrieved from http://mwexpert.typepad.com/rog_blog/2010/11/what-is-outgassing-and-when-does-it-matter.html.

[12] S. Sutton, �Measurement of cell concentration in sus-pension by optical density.� (August, 2006). Retrievedfrom http://www.microbiol.org/resources/monographswhite-papers/measurement-of-cell-concentration-in-suspension-by-optical-density/.

[13] D. Barich, �Thermus thermophilus.� (2010-11-16). Retrieved fromhttps://microbewiki.kenyon.edu/index.php/Thermus_thermophilus.

[14] J. B. Microbiol., �Growth of escherichia coli at elevated temperatures.� (2005).Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/16187264.

[15] J. D. van Elsas, �Survival of escherichia coli in the environment: fun-damental and public health aspects.� (2010-06-24). Retrieved fromhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105702/.

[16] M. T. Inc., �Mcp9700 series datasheet.� (2016). Retrieved fromhttp://www.digikey.se/product-search/en?keywords=mcp9700a-e.

[17] F. S. Inc., �Mpx5100 series datasheet.� (2010-05-13). Retrieved fromhttp://www.digikey.se/product-search/en?keywords=MPXV5100DP.

[18] V. Burt, �A few guidelines for selecting �lters.� (July 2005). Retrieved fromhttp://medicaldesign.com/components/few-guidelines-selecting-�lters.

50

IntroductionAim of ProjectBacteria in SpaceLife Support Systems and Pocket Earth EcosystemsDetecting Bacteria GrowthStudies of Previous Work

BackgroundThe MIST satellite projectMOREBACThe Employer

TheoryExperiment LimitationsSerial CommunicationExperimental Preparations and ProcedureExperiment Protocol

Design, Construction and TestingElectrical ComponentsMicrocontroller and Computer SoftwaresOptical Measurement ComponentsTemperature and Pressure Sensor CalibrationChoosing Resistances

Chip designOptical Measurement ConfigurationArduino and Processing programmingDye and Bacteria MeasurementsExperiment Protocol Testing

ResultsResistance testsAnalytical ResistancesTypical Photoresistance Measurements

Dye and Bacteria measurementsTemperature and Pressure CalibrationExperiment ProtocolLogged EventsReal Time Monitoring Graph

DiscussionConclusionFuture workReferences

Construction, programming and testing of measurement ...1078863/FULLTEXT01.pdf · bacteria growth,...

Documents

Transcript of Construction, programming and testing of measurement ...1078863/FULLTEXT01.pdf · bacteria growth,...